Antibodies to Mch6 polypeptides

ABSTRACT

The invention provides an isolated gene encoding Mch6 as well as functional fragments thereof. Also provided are isolated nucleic acid sequences encoding Mch6 or functional fragments thereof. The gene or nucleic acid sequences can be single or double stranded nucleic acids corresponding to coding or non-coding strands of the Mch6 nucleotide sequences. The invention further provides an isolated Mch6 polypeptide and isolated large and small subunits of the Mch6 polypeptide, including functional fragments thereof.

This application is a continuation of 09/746,731, filed Dec. 22, 2000,which application is a continuation of U.S. application Ser. No.09/257,218, filed Feb. 25, 1999, which was issued Aug. 7, 2001 as U.S.Pat. No. 6,271,361; which application is a divisional of U.S.application Ser. No. 08/865,579, filed May 29, 1997, which was issuedSep. 24, 2002 as U.S. Pat. No. 6,455,296; all applications are hereinincorporated by reference.

This invention was made with government support under research grant AI35035 awarded by the National Institutes of Health. The government hascertain rights in the invention.

BACKGROUND OF THE INVENTION

The present invention relates generally to apoptosis, or programmed celldeath, and more particularly, to a novel aspartate-specific cysteineprotease that can be used to modulate apoptosis for the therapeutictreatment of human diseases.

Apoptosis is a normal physiological process of cell death that plays acritical role in regulating tissue homeostasis by ensuring that the rateof new cell accumulation produced by cell division is offset by acommensurate rate of cell loss due to cell death. It has now becomeclear that disturbances in apoptosis, also referred to as physiologicalcell death or programmed cell death, which prevent or delay normal cellturnover, can be just as important to the pathogenesis of diseases asknown abnormalities in the regulation of proliferation and the cellcycle. Like cell division, which is controlled through complexinteractions between cell cycle regulatory proteins, apoptosis issimilarly regulated under normal circumstances by the interaction ofgene products that either induce or inhibit cell death.

The stimuli that regulate the function of these apoptotic gene productsinclude both extracellular and intracellular signals. Either thepresence or the removal of a particular stimulus can be sufficient toevoke a positive or negative apoptotic signal. For example,physiological stimuli that prevent or inhibit apoptosis include, forexample, growth factors, extracellular matrix, CD40 ligand, viral geneproducts, neutral amino acids, zinc, estrogen and androgens. Incontrast, stimuli that promote apoptosis include growth factors such astumor necrosis factor (TNF), Fas, and transforming growth factor β(TGFβ). Other stimuli that promote apoptosis include, for example,neurotransmitters, growth factor withdrawal, loss of extracellularmatrix attachment, intracellular calcium and glucocorticoids. Otherstimuli, including those of environmental and pathogenetic origins, caneither induce or inhibit programmed cell death. Although apoptosis ismediated by diverse signals and complex interactions of cellular geneproducts, the results of these interactions ultimately feed into a celldeath pathway that is evolutionarily conserved between humans andinvertebrates.

Several gene products that modulate the apoptotic process have now beenidentified. Although these products can be generally separated into twobasic categories, gene products from each category can function toeither inhibit or induce programmed cell death. One family of geneproducts is related to the protein Bcl-2, which inhibits apoptosis whenoverexpressed in cells. Other members of this gene family include, forexample, Bax, Bak, Bcl-x_(L), Bcl-x_(S), and Bad. While some of theseproteins can prevent apoptosis, others augment apoptosis, for example,Bcl-x_(S) and Bak, respectively.

A second family of gene products, the aspartate-specific cysteineproteases (ASCPs), are genetically related to the ced-3 gene product,which was initially shown to be required for programmed cell death inthe roundworm, C. elegans. The ASCP family of proteases includes humanICE (interleukin-1-β converting enzyme), ICH-_(L), ICH-1_(S), CPP32,Mch2, Mch3, Mch4, Mch5, ICH-2 and ICE_(rel)-III. Among the commonfeatures of these gene products are that 1) they are cysteine proteaseswith specificity for substrate cleavage at Asp-x bonds, 2) they share arelatively conserved pentapeptide sequence, QACRG (SEQ ID NO: 79) orQACQG (SEQ ID NO: 80), within the active site and 3) they aresynthesized as proenzymes that require proteolytic cleavage at specificaspartate residues for activation of protease activity. In the case ofICE, cleavage of the proenzyme produces two polypeptide proteasesubunits of approximately 20 kDa, known as p20, and 10 kDa, known asp10, that combine non-covalently to form a tetramer comprising twop20:p10 heterodimers. Although these proteases induce cell death whenexpressed in cells, several alternative structural forms, such as ICEδ,ICEε, ICH-1_(S) and Mch2β, actually function to inhibit apoptosis.

In addition to the Bcl-2 and ASCP gene families that play a role inapoptosis in mammalian cells, it has become increasingly apparent thatother gene products that are important in mammalian cell death have yetto be identified. For example, in addition to Ced-3, another C. elegansgene known as Ced-4 is also required for programmed cell death in C.elegans. However, mammalian homologs of Ced-4 remain elusive and havenot yet been identified. Further, it is ambiguous whether other genesbelong to either of the above two apoptotic gene families or what rolethey may play in the programmed cell death pathway. Finally, it isunclear what physiological control mechanisms regulate programmed celldeath or how the cell death pathways interact with other physiologicalprocesses within the organism. For example, it has recently beensuggested that cytotoxic T-lymphocytes mediate their destructivefunction by inducing apoptosis in their target cells.

Apoptosis maintains tissue homeostasis in a range of physiologicalprocesses such as embryonic development, immune cell regulation andnormal cellular turnover. Therefore, the dysfunction or loss ofregulated apoptosis can lead to a variety of pathological diseasestates. For example, the loss of apoptosis can lead to the pathologicalaccumulation of self-reactive lymphocytes such as that occurring withmany autoimmune diseases. Inappropriate loss of apoptosis can also leadto the accumulation of virally infected cells and of hyperproliferativecells such as neoplastic or tumor cells. Similarly, the inappropriateactivation of apoptosis can also contribute to a variety of pathologicaldisease states including, for example, acquired immunodeficiencysyndrome (AIDS), neurodegenerative diseases and ischemic injury.Treatments that are specifically designed to modulate the apoptoticpathways in these and other pathological conditions can change thenatural progression of many of these diseases.

Thus, there exists a need to identify new apoptotic genes and their geneproducts to modulate apoptosis for the therapeutic treatment of humandiseases. The present invention satisfies this need and provides relatedadvantages as well.

SUMMARY OF THE INVENTION

The invention provides an isolated gene encoding Mch6 as well asfunctional fragments thereof. Also provided are isolated nucleic acidsequences encoding Mch6 or functional fragments thereof. The gene ornucleic acid sequences can be single or double stranded nucleic acidscorresponding to coding or non-coding strands of the Mch6 nucleotidesequences. The invention further provides an isolated Mch6 polypeptideand isolated large and small subunits of the Mch6 polypeptide, includingfunctional fragments thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1a, 1 b and 1 c show the nucleotide and predicted amino acidsequence of Mch6, listed as SEQ ID NO: 1 and SEQ ID NO: 2, respectively.The active site pentapeptide sequence QACGG (SEQ ID NO: 78) isunderlined. Cleavage sites after Asp315 and Asp330 are indicated byvertical arrows.

FIG. 2 shows a multiple amino acid sequence alignment of relativelyconserved regions within the ASCPs. The ASCPs are Mch6 (SEQ ID NO: 6,consisting of noncontiguous SEQ ID NOs: 17-22), Mch5 (SEQ ID NO: 7,consisting of noncontiguous SEQ ID NOs: 23-27), Mch4 (SEQ ID NO: 8,consisting of noncontiguous SEQ ID NOs: 28-32), Mch3 (SEQ ID NO: 9,consisting of noncontiguous SEQ ID NOs: 33-37), Mch2 (SEQ ID NO: 10,consisting of noncontiguous SEQ ID NOs: 38-43), CPP32 (SEQ ID NO: 11,consisting of noncontiguous SEQ ID NOs: 44-48), CED-3 (SEQ ID NO: 12,consisting of noncontiguous SEQ ID NOs: 49-53), ICE (SEQ ID NO: 13,consisting of noncontiguous SEQ ID NOs: 54-59), TX (SEQ ID NO: 14,consisting of noncontiguous SEQ ID NOs: 60-65), ICErelIII (SEQ ID NO:15, consisting of noncontiguous SEQ ID NOs: 66-71) and ICH-1 (SEQ ID NO:16, consisting of noncontiguous SEQ ID NOs: 72-77).

Based on the crystal structure of ICE, specific residues are indicatedby lowercase letters below the sequences: “c” for residues involved incatalysis, “b” for residues that bind the substrate-carboxylate of P1Asp and “a” for residues adjacent to the substrate P2-P4 amino acids.“DX” indicates known and potential processing sites between the smalland large subunits of ASCPs. The roman numerals on the left indicate thethree ASCP-subfamilies: the Ced-like subfamily (I), the ICE-likesubfamily (II) and the Nedd2/Ich-1 subfamily (III). The asteriskindicates the nonconservative substitution in the active sitepentapeptide sequences of Mch4, Mch5 and Mch6.

FIG. 3 shows a schematic diagram illustrating the processing of proMch6.proMch6 can be processed by CPP32 after Asp330. proMch6 can also beprocessed preferentially after Asp315 by granzyme B to generate thelarge subunit, known as p35, and the small subunit, known as p10, ofmature Mch6. The active site pentapeptide QACGG (SEQ ID NO: 78) in thelarge subunit is indicated.

DETAILED DESCRIPTION OF THE INVENTION

This invention is directed to a novel apoptotic protease termed Mch6(mammalian ced-3 homolog 6). Mch6 is a member of the aspartate-specificcysteine protease (ASCP) family of proteases that includes, for example,ICE (Alnemri et al., J. Biol. Chem. 270:4312-4317 (1995)), CPP32(Fernandes-Alnemri et al., J. Biol. Chem. 269:30761-30764 (1994)),Nedd2/Ich-1 (Kumar et al., Genes & Development 8:1613-1626 (1994); Wanget al., Cell 78:739-750 (1994)), Mch2 (Fernandes-Alnemri et al., CancerRes. 55:2737-2742 (1995)), Mch3 (Fernandes-Alnemri et al., Cancer Res.55:6045-6052 (1995), Mch4 (Fernandes-Alnemri et al., Proc. Natl. Acad.Sci. USA 93:7464-7470 (1996)), Mch5 (Fernandes-Alnemri et al. (1996)supra), TX (ICH-2, ICErel-II) (Faucheu et al., EMBO 14:1914-1922 (1995);Kamens et al., J. Biol. Chem. 270:15250-15256 (1995); Munday et al., J.Biol. Chem. 270:15870-15876 (1995)) and ICErel-III (Munday et al. (1995)supra).

Mch6 shares amino acid sequence homology with several ASCPs, but itscatalytic site QACGG (SEQ ID NO: 78) differs in the fourth residue fromthe relatively conserved catalytic sites in other known ACSPs (FIGS. 1and 2).

Like many ASCPs, Mch6 is synthesized as a proenzyme, which can beproteolytically cleaved by, for example, CPP32 or granzyme B. Thecleavage of Mch6 by these two enzymes is described further below inExamples III and IV, respectively. Cleavage of Mch6 yields two subunits,a large subunit of approximately 35 kDa and a small subunit ofapproximately 10 kDa, which associate to form an active heterodimercomplex. Like other ASCPs, the active Mch6 complex can act as a proteaseand requires an Asp residue in the P1 position of the substrate bindingsite with a small, preferably hydrophobic, residue in the P1′ position.

In one embodiment, the invention is directed to nucleic acids encodingthe apoptotic protease Mch6. The nucleic acids are used to produce therecombinant Mch6 ASCP protease. The recombinant polypeptides can be usedto screen for Mch6 inhibitors. Mch6 inhibitors include those thatinhibit protease activity as well as compounds that inhibit Mch6 bindingto other polypeptides. Such compounds are useful as pharmaceuticals fortreating or preventing diseases characterized by apoptotic cell death.Alternatively, the Mch6 polypeptides can be used to screen for compoundsthat activate or act as agonists of Mch6, such as by inducing cleavageof the proenzyme into its active subunits. Such compounds are similarlyuseful as pharmaceuticals for treating or preventing diseasescharacterized by the loss of apoptotic cell death.

As used herein, the term “substantially” when referring to a Mch6nucleotide or amino acid sequence is intended to refer to the degree towhich two sequences of between about 15-30 or more nucleotides in lengthare identical or similar, so as to be considered by those skilled in theart to be functionally equivalent. For example, the Mch6 nucleic acid ofthe invention has a nucleotide sequence substantially the same as thatshown in FIG. 1 and as SEQ ID NO: 1. Thus, if a second sequence isconsidered by those skilled in the art to be functionally equivalent tothe sequence shown as SEQ ID NO: 1, then the second sequence issubstantially the same as that shown as SEQ ID NO: 1. Methods forsequence comparisons and determinations of similarity are well known androutine within the art.

Functionally equivalent nucleic acid sequences include, for example,sequences that are related, but different and encode the same Mch6polypeptide due to the degeneracy of the genetic code as well assequences that are related, but different and encode a different Mch6polypeptide that exhibits similar functional activity. In both cases,the nucleic acids encode functionally equivalent gene products.Functional fragments of Mch6 encoding nucleic acids such asoligonucleotides, polynucleotides, primers and the like are alsoconsidered to be within the definition of the term and the invention asclaimed. Functional equivalency is also relevant to Mch6 nucleic acidsthat do not encode gene products, for example, but instead arefunctional elements in and of themselves. Specific examples of suchfunctional nucleic acids include, for example, promoters, enhancers andother gene expression and transcription regulatory elements.

An Mch6 polypeptide of the invention has an amino acid sequencesubstantially similar to that shown in FIG. 1 and in SEQ ID NO: 2.Functionally equivalent Mch6 amino acid sequences similarly includes,for example, related, but different sequences so long as the differentpolypeptide exhibits at least one functional activity of Mch6. Suchrelated, but different polypeptides include, for example, substitutionsof conserved and nonessential amino acids. Fragments and functionaldomains of Mch6 are similarly included within the definition of the termand the claimed invention.

Therefore, it is understood that limited modifications may be madewithout destroying the biological function of the Mch6 polypeptide andthat only a portion of the entire primary structure may be required inorder to effect activity. For example, minor modifications of the Mch6amino acid sequence (SEQ ID NO: 2) that do not destroy their activityalso fall within the definition of Mch6 and within the definition of thepolypeptide claimed as such. Also within the definition of the claimedpolypeptides are, for example, genetically engineered fragments of Mch6,either alone or fused to heterologous proteins such as fusion proteinsthat retain measurable enzymatic or other biological activity.

It is understood that minor modifications of primary amino acid sequencemay result in polypeptides that have substantially equivalent orenhanced function as compared to the sequences set forth in FIG. 1 (SEQID NO: 2). These modifications may be deliberate, as throughsite-directed mutagenesis, or may be accidental such as through mutationin hosts that are Mch6 producers. All of these modifications areincluded as long as Mch6 biological function is retained. Further,various molecules can be attached to Mch6, for example, other proteins,carbohydrates, lipids, or chemical moieties. Such modifications areincluded within the definition of an Mch6 polypeptide.

The invention provides a gene encoding Mch6, or fragment thereof. Theinvention also provides an isolated nucleic acid sequence encoding Mch6,or fragment thereof. The gene and nucleic acid sequences encodesubstantially the sequence as shown in SEQ ID NO: 1. Fragments of thegene or nucleic acid sequence are provided that comprise single ordouble stranded nucleic acids having substantially the sequences shownin SEQ ID NO: 1.

The Mch6 nucleic acid of the present invention was identified andisolated by a novel approach of searching a human database of expressedsequence tags (ESTs) under various stringencies to identify potentiallynew sequence fragments that may have homology to the ICE family ofcysteine proteases. Previously these proteases were referred to as theICE-family of proteases and thus the initial search criteria wasdirected to the ICE family of cell death proteases. However, with therecent identification of Mch4 and Mch5, the proteases have beenreclassified into three subfamilies referred to herein as the Ced-like,ICE-like and Nedd2/ICH-1-like subfamilies of cell death proteases.

When searching for potential new sequences related to the ICE family ofproteases, novel sequences are identified by their homology to the ICEfamily of cell death proteases. These novel sequences are then used todesign primers for attempting PCR amplification and cloning of theactual cDNA. The second primer for the amplification is designed toencompass homologous regions in nucleic acid sequences that encode knownICE protease family members. In this specific case, the primer wasdirected to a sequence analogous to the GSWFI/GSWYI pentapeptidesequence that is conserved in a number of the ICE/Ced-3 family ofproteases. The primer design should take into account the predictedstrandedness of both the EST sequence primer and the known primer. Thus,only if the homology search and primer hybridization conditions aresuccessfully determined will such an approach allow PCR amplification ofa fragment of the putative novel protease cDNA.

Because searching a genetic data base will yield homologous sequencematches to any nucleotide sequence query, additional criteria must beused to identify the authentic ICE subfamily homolog from among thenon-specific homology matches. ICE family members share the highestdegree of homology in the active site and catalytically important aminoacid residues. A given EST returned by the search may not include one ofthese highly homologous sites, but may only include a region within theprotease with cryptic homology. Confirming an EST as a novel ICEprotease involves translation of all the positive EST hits in threedifferent reading frames and subsequent identification of conservativeactive site or catalytically important amino acid sequence motifs. Then,using conventional cDNA cloning, a full length cDNA of the putativenovel protease can be obtained and 1) analyzed for overall structuralhomology to ICE family members, 2) recombinantly expressed and analyzedfor cysteine protease activity, and 3) analyzed for the induction ofprogrammed cell death by heterologous expression of the cDNA inappropriate cells.

In addition to the methods described above for isolating Mch6 encodingnucleic acids, alternative methods can similarly be employed. Forexample, using the teachings described herein, those skilled in the artcan routinely isolate and manipulate Mch6 nucleic acids using methodswell known in the art. All that is necessary is the sequence of theMch6-encoding nucleic acid (FIG. 1 and SEQ ID NO: 1) or its amino acidsequence (FIG. 1 and SEQ ID NO: 2). Such methods include, for example,screening a cDNA or genomic library by using synthetic oligonucleotides,nucleic acid fragments or primers as hybridization probes.Alternatively, antibodies to the Mch6 amino acid sequence or fragmentsthereof can be generated and used to screen an expression library toisolate Mch6-encoding nucleic acids. Other binding reagents to an Mch6polypeptide can similarly be used to isolate an Mch6 polypeptide havingsubstantially the amino acid sequence shown in FIG. 1. Similarly,substrate reagents such as non-cleavable peptide analogs of cysteineproteases can be used to screen and isolate an Mch6 polypeptide.

In addition, recombinant DNA methods currently used by those skilled inthe art include-the polymerase chain reaction (PCR). When combined withthe Mch6 nucleotide and amino acid sequences described herein, PCRallows reproduction of Mch6 encoding sequences. PCR can amplify desiredsequences exponentially starting from as little as a single gene copy.The PCR technology is the subject matter of U.S. Pat. Nos. 4,683,195,4,800,159, 4,754,065 and 4,683,202, all of which are incorporated byreference herein.

The above-described methods are known to those skilled in the art andare described, for example, in Sambrook et al., Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory, NY (1992) and thevarious references cited therein and in Ansubel et al., CurrentProtocols in Molecular Biology, John Wiley and Sons, Baltimore, Md.(1989); and in Harlow et al., Antibodies: A Laboratory Manual, ColdSpring Harbor Laboratory, NY(1989). These references and thepublications cited therein are hereby expressly incorporated herein byreference.

The invention provides an isolated Mch6 polypeptide comprisingsubstantially the amino acid sequence as that shown in FIG. 1 (SEQ IDNO: 2). Mch6 functional fragments are also provided. Specific examplesof Mch6 functional fragment include, for example, the catalytic domainthat contains the active site amino acid sequence QACGG (SEQ ID NO: 78).When compared to the active site amino acid sequence of other ASCPfamily members, QACRG (SEQ ID NO: 79) or QACQG (SEQ ID NO: 80), thisactive site sequence is similar but differs at position 4 with Arg (R)substituted by Gly (G).

An isolated Mch6 polypeptide of the invention can be obtained by avariety of methods known within the art. For example, the isolatedpeptides can be purified by biochemical methods including affinitychromatography, for example. Affinity matrices for Mch6 isolation can beanti-Mch6 monoclonal or polyclonal antibodies prepared against the aminoacid sequence shown in FIG. 1 (SEQ ID NO: 2) or against fragmentsthereof such as synthetic peptides. Alternatively, substrate analoguesor enzymatic inhibitors of Mch6 can similarly be used as affinitymatrices to isolate substantially pure a Mch6 polypeptide of theinvention.

An Mch6 polypeptide can also be produced by recombinant methods known tothose skilled in the art. Recombinant Mch6 polypeptides include, forexample, an amino acid sequence substantially the same as that shown inFIG. 1 (SEQ ID NO: 2), as well as fusion proteins and fragments thereof.The Mch6-encoding nucleic acids can be cloned into the appropriatevectors for propagation, manipulation and expression. Such vectors areknown or can be constructed by those skilled in the art and shouldcontain all expression elements necessary for the transcription,translation, regulation, and if desired, sorting of the Mch6polypeptides. The vectors can also be for use in either procaryotic oreucaryotic host systems so long as the expression and regulatoryelements are compatible. One of ordinary skill in the art will knowwhich host systems are compatible with a particular vector. Therecombinant polypeptides produced can be isolated by the methodsdescribed above.

The invention further provides isolated large and small subunits of anMch6 polypeptide. For example, the proMch6 polypeptide can beproteolytically cleaved by CPP32 or granzyme B to form a large and smallsubunit (Examples III and IV). In particular, CPP32 can cleave proMch6into a large subunit having an approximate molecular weight of 37 kDa(p37) and a small subunit having an approximate molecular weight of 10kDa (p10). Similarly, granzyme B can cleave proMch6 into a large subunithaving an approximate molecular weight of 35 kDa (p35) and a smallsubunit having an approximate molecular weight of 12 kDa (p12).Moreover, other components of the apoptotic pathway can process Mch6into a larger and a smaller cleavage product. Accordingly, the terms“large subunit” and “small subunit” will readily be understood to referto any larger proteolytic cleavage product such as p37 or p35, and anysmaller cleavage product such as p10 or 12, respectively.

Apoptosis plays a significant role in numerous pathological conditionsin that programed cell death is either inhibited, resulting in increasedcell survival, or enhanced, which results in the loss of cell viability.Examples of pathological conditions resulting from increased cellsurvival include cancers such as lymphomas, carcinomas andhormone-dependent tumors. Such hormone-dependent tumors include, forexample, breast, prostate and ovarian cancer. Increased cell survival orapoptosis inhibition can also result in autoimmune diseases such assystemic lupus erythematosus and immune-mediated glomerulonephritis, aswell as viral infections such as herpesvirus, poxvirus and adenovirus.

In contrast, apoptotic diseases where enhanced programed cell death is aprevalent cause generally includes, for example, degenerative disorderssuch as Alzheimer's disease, Parkinson's disease, amyotrophic lateralsclerosis, retinitis pigmentosa, and cerebellar degeneration. Otherdiseases associated with increased apoptosis include, for example,myelodysplastic syndromes such as aplastic anemia and ischemic injury,including myocardial infarction, stroke and reperfusion injury.

The Mch6-encoding nucleic acids and polypeptides of the invention can beused to diagnose, treat or reduce the severity of cell death-mediateddiseases such as those described above as well as other diseasesmediated by either increased or decreased programmed cell death.Additionally, the Mch6-encoding nucleic acids and polypeptides of theinvention can be used to screen for pharmaceutical compounds andmacromolecules that inhibit or promote Mch6-mediated apoptosis.

For example, the Mch6-encoding nucleic acids, polypeptides andfunctional fragments thereof can be used to diagnose or generatereagents to diagnose diseases mediated or characterized by programedcell death. Diagnosis can be by nucleic acid probe hybridization withMch6-containing nucleotide sequences, antibody- or ligand-mediateddetection with Mch6-binding agents, or by enzyme catalysis of detectableMch6 substrates. Such methods are routine to those skilled in the art.Detection can be performed ex vivo, for example, by removing a cell ortissue sample from an individual exhibiting or suspected of exhibiting acell death-mediated disease. Correlation of increased Mch6 expression oractivity is indicative of diseases characterized by enhanced programmedcell death, whereas correlation of decreased Mch6 expression or activityis indicative of diseases characterized by the inhibition of programmedcell death.

The above Mch6 polypeptide can also be formulated into pharmaceuticalcompositions for treating cell death-mediated diseases characterized byincreased cell survival and proliferation. Functional fragments andpeptides such as the catalytic domain of Mch6 can similarly beformulated to treat such diseases. Additionally, molecules that interactwith Mch6 can also be used to induce Mch6-mediated apoptosis.Administration of Mch6 polypeptides and functional fragments thereofwill induce apoptosis in treated cells and eliminate those cellscharacterized by increased cell survival or proliferation. Non-Mch6polypeptides that do not directly act on Mch6 substrates but induce theactivation of the Mch6 protease can similarly be administered to treatdiseases characterized by increased cell survival and proliferation.

To be effective, the Mch6 polypeptide must be introduced into the cellsby means characterized by increased cell survival. Introduction can beaccomplished by a variety of means known within the art including, forexample, lipid vesicles and receptor-mediated endocytosis. Targeting tothe appropriate cell type can similarly be accomplished throughconjugation to specific receptor ligands, specific target cellantibodies and the like.

The Mch6 polypeptide is administered by conventional methods in dosagesthat are sufficient to induce apoptosis in the cells characterized byincreased cell survival or proliferation. Such dosages are known or canbe easily determined by those skilled in the art. Administration can beaccomplished, for example, by intravenous, interperitoneal orsubcutaneous injection. Administration can be performed in a variety ofdifferent regimes that include single high dose administration, repeatedsmall dose administration, or a combination of both. The administrationwill depend on the cell type, progression of the disease and overallhealth of the individual and will be known or can be determined by thoseskilled in the art.

In contrast to the induction of Mch6-mediated apoptosis to treatpathological conditions characterized by increased cell survival orproliferation, inhibitors of Mch6 can be used to treat diseasescharacterized by increased programmed cell death. Mch6 inhibitorsinclude, for example, small molecules and organic compounds that bindand inactivate Mch6 protease activity by a competitive ornoncompetitive-type mechanism, inhibitors of the conversion of inactiveproMch6 into active Mch6 protease or other molecules that indirectlyinhibit the Mch6 pathway. Such Mch6 inhibitors can include, for example,suicide inhibitors, anti-Mch6 antibodies and proteins, small peptidylprotease inhibitors, or small non-peptide organic molecule inhibitors.Specific examples of such inhibitors are described in Example II, andinclude substrate analogs such as tetrapeptide DEVD-CHO(Asp-Glu-Val-Asp-aldehyde; SEQ ID NO: 81), DEVD-AMC(Asp-Glu-Val-Asp-aminomethylcoumarin; SEQ ID NO: 82), YVAD-AMC(Tyr-Val-Ala-Asp-aminomethylcoumarin; SEQ ID NO: 83), ZEVD-AMC(carbobenzoxy-Glu-Val-Asp-aminomethylcoumarin) and the cowpox virusprotein Crm A. Mch6 inhibitors can be formulated in a medium that allowsintroduction into the desired cell type or can be attached to targetingligands for introduction by cell-mediated endocytosis and otherreceptor-mediated events.

Mch6 inhibitors can also be used to treat or reduce the severity ofdiseases characterized by increased programmed cell death. In thisregard, Mch6 large subunits that lack the active site QACGG (SEQ ID NO:78) can be used to bind the small subunit of Mch6 to prevent theformation of active protease complexes. Such a mechanism of dominantnegative inhibition of Mch6 is similar to the dominant negativeinhibition of Ich-1_(L) by Ich-1_(S). Subunits from other ASCPs cansimilarly be used as dominant/negative inhibitors of Mch6 activity andtherefore be used to treat diseases mediated by programmed cell death.Such subunits should be selected so they bind either the p35 or p10 Mch6polypeptide and prevent their assembly into active tetrameric proteasecomplexes. Moreover, Mch6 subunits that have been modified to becatalytically inactive can also be used as dominant negative inhibitorsof Mch6. Such modifications include, for example, mutation of the activesite cysteine residue to include alanine or glycine, for example.

Mch6 substrate antagonists can similarly be used to treat or reduce theseverity of diseases mediated by increased programmed cell death. Suchsubstrate antagonists can bind to and inhibit cleavage by Mch6.Inhibition of substrate cleavage prevents commitment progression ofprogrammed cell death. Substrate antagonists include, for example,ligands and small molecule compounds

Mch6 inhibitors can be identified by incubating cells with a compound tobe tested for inhibitory activity and then treated with a stimulus ofapoptosis such as anti-Fas antibody, Fas ligand or staurosporin. Controlsamples in the absence of inhibitors that are subsequently treated withanti-Fas antibody undergo apoptosis and exhibit a rapid induction ofASCP activity. In contrast, samples in the presence of inhibitors reduceor negate ASCP activity compared to that in control samples.

Mch6 inhibitors can also be identified using Mch6-encoding nucleic acidsand the Mch6 polypeptide of the invention in, for example, bindingassays such as ELISA or RIA, or enzymatic assays using tetrapeptidesubstrates, such as DEVD-AMC (SEQ ID NO: 82) and YVAD-AMC (SEQ ID NO:83). DEVD-AMC (SEQ ID NO: 82) and YVAD-AMC (SEQ ID NO: 83) representcleavage sites for the poly(ADP-ribose) polymerase (PARP) and IL-1βP1-P4substrate tetrapeptides, respectively (Nicholson et al., Nature376:37-43 (1995)).

The Mch6 polypeptide to be used in such assays can be obtained by, forexample, in vitro translation, recombinant expression or biochemicalprocedures. Such and other methods are known within the art, and areillustrated in Example II. For example, recombinant Mch6 can beexpressed by cloning Mch6 cDNA into a bacterial expression vector suchas pET21b (Novagen Inc., Madison, Wis.). The Mch6 can then be expressedand purified using routine molecular biology methods known to thoseskilled in the art. A purified recombinant Mch6 protein can be used tomeasure hydrolysis rates for various substrates, such as DEVD-AMC (SEQID NO: 82) and YVAD-AMC in a continuous fluorometric assay.

Using such an assay for Mch6 activity, various compounds can be screenedfor compounds that inhibit or enhance the expression of Mch6 proteaseactivity. Such screening methods are known to those skilled in the artand can be performed by either in vitro or in vivo procedures. Suchinhibitory molecules can be those contained in synthetic or naturallyoccurring compound libraries. A specific example is phage displaypeptide libraries where greater than 10⁸ peptide sequences can bescreened in a single round of panning.

Treatment or reduction of the severity of cell death-mediated diseasescan also be accomplished by introducing expressible nucleic acidsencoding a Mch6 polypeptide or functional fragments thereof into cellscharacterized by such diseases. For example, elevated synthesis rates ofMch6 can be achieved by, for example, using recombinant expressionvectors and gene transfer technology. Similarly, treatment or reductionof the severity of cell death-mediated diseases can also be accomplishedby introducing and expressing antisense Mch6 nucleic acids to inhibitthe Mch6 synthesis rate. Such methods are well known within the art andare described below with reference to recombinant viral vectors. Othervectors compatible with the appropriate targeted cell can accomplish thesame goal and therefore can be substituted in the methods describedherein in place of recombinant viral vectors.

Recombinant viral vectors are useful for in vivo expression of a desirednucleic acid because they offer advantages such as lateral infection andtargeting specificity. Lateral infection is inherent in the life cycleof retroviruses and is the process by which a single infected cellproduces many progeny virions that bud off and infect neighboring cells.The result is the rapid infection of a large area, most of which was notinitially infected by the original viral particles. This is in contrastto vertical-type infection in which the infectious agent spreads onlythrough daughter progeny. Viral vectors can also be produced that areunable to spread laterally. This characteristic can be useful if thedesired purpose is to introduce a specified gene into only a localizednumber of targeted cells.

Typically, viruses infect and propagate in specific cell types.Therefore, the targeting specificity of viral vectors utilizes thisnatural specificity to specifically introduce a desired gene intopredetermined cell types. The vector to be used in the methods of theinvention will depend on desired cell type to be targeted. For example,to treat neurodegenerative diseases by decreasing the Mch6 activity ofaffected neuronal cells, a vector should be used that is specific forcells of the neuronal cell lineage. Likewise, to treat diseases orpathological conditions of hematopoietic cells, a viral vector should beused that is specific for blood cells and their precursors, preferablyfor the specific type of hematopoietic cell. Moreover, such vectors canbe modified with specific receptors or ligands and the like to modify oralter target specificity through receptor-mediated events. Thesemodification procedures can be performed by, for example, recombinantDNA techniques or synthetic chemistry procedures. The specific type ofvector will depend upon the intended application. The actual vectors arealso known and readily available within the art or can be constructed byone skilled in the art using well known methodology.

Viral vectors encoding Mch6 nucleic acids or inhibitors of Mch6 such asantisense nucleic acids can be administered in several ways to obtainexpression of such sequences and therefore either increase or decreasethe activity of Mch6 in the cells affected by the disease orpathological condition. If viral vectors are used, for example, theprocedure can take advantage of their target specificity andconsequently need not be administered locally at the diseased site.However, local administration can provide a quicker and more effectivetreatment. Administration can also be performed by, for example,intravenous or subcutaneous injection into the subject. Injection of theviral vectors into the spinal fluid can also be used as a mode ofadministration, especially in the case of neurodegenerative diseases.Following injection, the viral vectors will circulate until theyrecognize host cells with the appropriate target specificity forinfection.

As described above, one mode of administration of Mch6-encoding vectorscan be by direct inoculation locally at the site of the disease orpathological condition. Local administration is advantageous becausethere is no dilution effect and therefore a smaller dose is can besufficient to achieve Mch6 expression in a majority of the targetedcells. Additionally, local inoculation can alleviate the targetingrequirement associated with other forms of administration because avector can be used that infects all cells in the inoculated area. Ifexpression is desired in only a specific subset of cells within theinoculated area, then promoter and expression elements that are specificfor the desired subset can be used to accomplish this goal. Suchnon-targeting vectors can be, for example, viral vectors, viral genomes,plasmids, phagemids and the like. Transfection vehicles such asliposomes can be used to introduce the non-viral vectors described aboveinto recipient cells within the inoculated area. Such transfectionvehicles are known by one skilled within the art. Alternatively,however, non-targeting vectors can be administered directly into atissue of any individual. Such methods are known within the art and aredescribed by, for example, Wolff et al. (Science 247:1465-1468 (1990)).

Additional features can be added to the vectors to ensure safety and/orenhance therapeutic efficacy. Such features include, for example,markers that can be used to negatively select against cells infectedwith the recombinant virus. An example of such a negative selectionmarker is the TK gene described above, which confers sensitivity to theantibiotic gancyclovir. Infection can therefore be controlled bynegative selection because it provides inducible suicide through theaddition of an antibiotic. Such protection ensures that if, for example,mutations arise that produce mutant forms of Mch6, dysfunction ofapoptosis will not occur.

It is understood that modifications that do not substantially affect theactivity of the various embodiments of this invention are also includedwithin the definition of the invention provided herein. Accordingly, thefollowing examples are intended to illustrate but not limit the presentinvention.

EXAMPLE I Cloning and Characterization of Mch6

This Example shows the cloning, sequence analysis and tissuedistribution of Mch6. The results described herein indicate that Mch6 isa novel member of the ICE family of aspartate-specific cysteineproteases (ASCPs).

To identify potentially novel members of the ICE family of ASCPs, anapproach combining information from the GenBank database of humanexpressed sequence tags (ESTs) and PCR was employed (seeFernandes-Alnemri et al., Cancer Res. 55:2737-2742 (1995);Fernandes-Alnemri et al., Cancer Res. 55:6045-6052 (1995)). Initially, asearch of GenBank ESTs for sequences related to CPP32 and Mch2identified a short EST sequence, which was used to derive a PCR primerto clone related cDNAs. The EST sequence was identified as accessionnumber T97582 and was used to design primer Mch6-prl, having thenucleotide sequence CTCAACGTACCAGGAGCC (SEQ ID NO: 3). A 10 μl aliquotof human Jurkat λ Uni-Zap™ XR cDNA library (Fernandes-Alnemri et al., J.Biol. Chem. 269:30761-30764 (1994)) containing approximately 10⁸ pfu wasdenatured at 99° C. for 5 min. and used as a template for PCRamplification with the above Mch6-pr1 primer and a T3 vector-specificprimer (Stratagene).

A 10 μl aliquot of the primary amplification product was then used as atemplate for a secondary PCR amplification with primer Mch6-pr2, havingnucleotide sequence CCTGGGAAAGTAGAGTAGG (SEQ ID NO: 4), which was alsoderived from EST sequence T97582, and the SK-Zap vector-specific primerlocated downstream of the T3 primer, having nucleotide sequenceCAGGAATTCGGCACGAG (SEQ ID NO: 5). The secondary amplification productswere cloned into a SmaI-cut pBluescript II KS⁺ vector. The partial cDNAwas then excised from the vector, radiolabeled and used to screen theoriginal Jurkat λ Uni-Zap™0 XR cDNA library for full length cDNA clones.Positive λ clones were purified, rescued into the pBluescript IISK⁻plasmid vector and sequenced.

The screen of the full length Jurkat λ Uni-Zap™ XR cDNA library resultedin the isolation of an approximately 2 kb cDNA clone. This cDNA containsan open reading frame of 1248 bp (SEQ ID NO: 1) that encodes a 416-aminoacid protein, named proMch6 (SEQ ID NO: 2). As shown in FIG. 1, proMch6is a polypeptide of 479 amino acid residues with a predicted molecularmass of approximately 46.2 kDa.

Shown in FIG. 2 is a multiple amino acid sequence alignment ofrelatively conserved regions within the ASCPs. These regions include,for example, the relatively conserved active site pentapeptide sequenceQACRG (SEQ ID NO: 79), residues involved in catalysis, residues thatbind the substrate-carboxylate of P1 Asp, residues adjacent to thesubstrate P2-P4 amino acids and the known and potential processing sitesbetween the small and large subunits. Sequence alignment of all knownASCPs revealed that Mch6 belongs to the Ced-3-like subfamily of ASCPs.Briefly, previously identified ASCPs can be divided phylogeneticallyinto three subfamilies. The Ced-3-like ASCP subfamily includes Ced-3,CPP32, Mch2, Mch3, Mch4 and Mch5 (noncontiguous SEQ ID NOs: 6-11). TheICE-like ASCP subfamily includes ICE, TX (noncontiguous ICH-2,ICErel-II, Mihl) and ICErelIII (noncontiguous SEQ ID NOs: 12-14). TheNEDD-like subfamily includes ICH-1 (noncontiguous SEQ ID NO: 15) and itsmouse counterpart NEDD2. Within the Ced-3-like subfamily, Mch6 shows thehighest homology to CPP32 (approximately 37% identity, 57% similarity).

Mch6 is also structurally similar to other ASCPs. The mature Mch6 couldbe derived from the precursor proenzyme by cleavage at highly conservedAsp residues (Asp315 and Asp330) located between the two subunits. Onedifference between this enzyme and other family members, however, isthat the fourth residue in its active site pentapeptide sequence QACGG(SEQ ID NO: 78) is a Gly instead of Arg or Gln (FIG. 1).

Study of the known crystal structure of ICE has revealed that His237,Gly238 and Cys285 are involved in catalysis, while Arg179, Gln283,Arg341 and Ser347 are involved in binding the carboxylate side chain ofthe substrate P1 aspartate (Walker et al., Cell 78:343-352 (1994);Wilson, et al., Nature 370:270-275 (1994)). All these residues areidentical in all family members except in Mch5, where there is a Ser toThr conservative substitution for the residue corresponding to Ser347 ofICE (FIG. 2). Another Ser to Thr conservative substitution can also beseen in Mch4 in the region corresponding to Ser236 of ICE, which is oneof the residues that participates in binding the substrate P2-P4residues. Other residues that might participate in binding the substrateP2-P4 residues are not widely conserved, suggesting that they maydetermine substrate specificity.

In addition to the above sequence analysis, further characterization ofMch6 was also performed by analyzing its tissue distribution. Thisanalysis was performed by RNA blot analysis of poly A⁺ RNA isolated fromdifferent human tissues (Fernandes-Alnemri et al., Cancer Res.55:6045-6052 (1995)). Briefly, tissue distribution analysis of Mch6 mRNAwas performed on northern blots prepared by Clontech (San Diego, Calif.)containing 2 μg/lane of poly A⁺ RNA from each tissue of origin. Aradioactive Mch6 riboprobe was prepared using Mch6 cDNA as a templatefor T7 RNA polymerase in the presence of [α³²p]ATP. The blots werehybridized, washed and then visualized by autoradiography.

The proMch6 riboprobe detected three major mRNA species (approximately1.0 kb, approximately 2.4 kb and approximately 4.4 kb) in most humantissues. Highest expression was seen in the heart, and moderateexpression in the liver, skeletal muscle and pancreas. Lowest expressionwas observed in the other tissues tested. The presence of multiple mRNAspecies has been observed with ICE and is suggestive of alternativesplicing or polyadenylation (Cerretti et al., Science 256:97-100(1992)).

To determine if Mch6 exhibits apoptotic activity, Sf9 baculovirus cellsare used. Briefly, Sf9 cells are infected with recombinant baculovirusesencoding full length Mch6, full length CPP32 or truncated variants ofMch6 or CPP32, separately or in various combinations. Cells are thenexamined microscopically for morphological signs of apoptosis such asblebbing of the cytoplasmic membrane, condensation of nuclear chromatinor release of small apoptotic bodies. In addition, the genomic DNA isexamined for internucleosomal DNA cleavage.

EXAMPLE II Kinetic Parameters of Mch6

This Example characterizes the protease activity and substratespecificity of the ASCP Mch6.

The kinetic properties of bacterially expressed recombinant Mch6 weredetermined using the tetrapeptide substrates DEVD-AMC (SEQ ID NO: 82),ZEVD-AMC and YVAD-AMC (SEQ ID NO: 83) in a continuous fluorometric assay(Table I). The DEVD-AMC (SEQ ID NO: 82) and ZEVD-AMC represent thecleavage site for poly(ADP-ribose)polymerase (PARP). The YVAD-AMC (SEQID NO: 83) represents the cleavage site for IL-1β P1-P4 substratetetrapeptide (Nicholson et al., Nature 376:37-43 (1995)). Briefly, Mch6cDNA was cloned in-frame into bacterial expression vector pET21b(Novagen Inc., Madison, Wis.). The expression vector was constructed andexpressed as a 6His-C terminal tagged protein in host bacterial strainBL21(DE3) using routine molecular biology methods known to those skilledin the art. After induction with IPTG, bacterial extracts were preparedfrom E. coli expressing the recombinant proteins. The extracts were thenpurified on a Ni⁺⁺ affinity column.

The purified Mch6 protein was then used for further enzymatic analyses.The activity of Mch6 was measured using bacterial lysates prepared withICE buffer (25 mM HEPES, 1 mM EDTA, 5mM DTT, 0.1% CHAPS, 10% sucrose, pH7.5) at room temperature (24-25° C.). The K_(i) of DEVD-CHO (SEQ ID NO:81) was determined from the hydrolysis rate of 50 μM DEVD-AMC (SEQ IDNO: 82) following a 30 min preincubation of the enzyme with DEVD-CHO(SEQ ID NO: 81).

TABLE I Kinetic Parameters of Mch6 Parameters Value K_(m) (DEVD-AMC) 10μM K_(i) (DEVD-CHO) <10 nm V_(max)/K_(m) (DEVD-AMC) 100 V_(max)/K_(m)(ZEVD-AMC) 2.7 V_(max)/K_(m) (YVAD-AMC) <0.1

As shown above in Table I, Mch6 is potently inhibited by the DEVD-CHOpeptide (K_(i)<10 nM). Mch6 also shows a greater than 1000-foldpreference for the CPP32-like substrate DEVD-AMC (SEQ ID NO: 82)compared to the ICE-like substrate YVAD-AMC (SEQ ID NO: 83), bycomparing V_(max)/K_(m) for each substrate. These kinetic parametersindicates that Mch6 is more related to the CPP32-like ASCPs than to theICE-like enzymes.

EXAMPLE III CPP32 Processes ProMch6 in Vitro

This example shows that CPP32 processes proMch6 at the Asp330 site toyield a large and a small cleavage product.

Wild type proMch6 contains a potential CPP32 cleavage site at the³²⁷DQLD-A³³¹ site (SEQ ID NO: 85), which is similar to the DEVD-G site(SEQ ID NO: 86) in PARP and the DVVD-N site (SEQ ID NO: 87) in proMch2(Nicholson et al., Nature 376:37-43(1995)). These similarities suggestthat CPP32 can also process proMch6 as well.

To provide proMch6 as a potential processing substrate, proMch6 cDNA wastranscribed and translated in vitro in the presence of ³⁵S-methionineusing coupled transcription/translation TNT kit according to themanufacturer's recommendations (Promega, Madison Wis.). Two microlitersof the translation reaction were incubated with purified enzymes(100-200 ng) or bacterial lysates in ICE buffer (25 mM HEPES, 1 mM EDTA,5 mM DTT, 0.1% CHAPS, 10% sucrose, pH 7.5), in a final volume of 10 μl.The reaction was incubated at 37° C. for 1-2 hours and the resultingtranslation products were analyzed by Tricine-SDS-PAGE andautoradiography.

To determine the ability of CPP32 to process proMch6, proMch6 from invitro translation was incubated for various times with purifiedrecombinant human CPP32 and then analyzed by Tricine-SDS-PAGE andautoradiography. The CPP32 was obtained by expression in bacteria,assayed for activity and purified on a Ni⁺² affinity resin(Fernandes-Alnemri et al., Cancer Res. 55:2737-2742; Fernandes-Alnemriet al., Cancer Res. 55:6045-6052 (1995)).

Analysis of the time course revealed that CPP32 cleaves proMch6 at onesite to generate two cleavage products of approximately 37 kDa or p37and approximately 10 kDa or p10. The sizes of the products wereconsistent with cleavage at the ³²⁷DQLD-A³³¹ site (SEQ ID NO: 85), whichcontains Asp330. Products were initially detected within the first 15minutes. Longer incubation resulted in decreased intensity of the fulllength 46 kDa band and increased intensity of the p37 and p10 products.Prolonged incubation did not result in significant processing of the p37product. This result indicates that CPP32 cleaves preferentially at onlyone site within proMch6.

The CPP32 processing site was confirmed by constructing and analyzingmutants of proMch6. Briefly, potential processing sites were mutated bysite-directed mutagenesis using overlapping PCR mutagenicoligonucleotides. The resulting PCR products were subcloned inpBluescript II KS⁺ vector under the T7 promoter and their sequences wereverified by DNA sequencing. In particular, Asp315 of proMch6 was mutatedto Ala for one mutant and Asp330 was mutated to Ala for another mutant.

As with the wild type proMch6, the Asp315 mutant was processed by CPP32to generate the p37 and p10 products. In contrast, CPP32 failed toprocess the Asp330 mutant. These results show that CPP32 processesproMch6 at the Asp330 site.

EXAMPLE IV Granzyme B Cleaves ProMch6 Preferentially at the PEPD-A Site(SEQ ID NO: 84)

This example shows that granzyme B cleaves proMch6 at two sites, with apreference for cleaving at the ³¹²PEPD-A³¹⁶ site (SEQ ID NO: 84).

Granzyme B can induce apoptosis in target cells by activation of ASCPs.The ability of granzyme B to process several members of the ASCP familyhas now been demonstrated (Fernandes-Alnemri et al., Proc. Natl. Acad.Sci. USA 93:7464-7469 (1996)). To test whether granzyme B can processproMch6, ³⁵S-labeled proMch6 was prepared and incubated with granzyme Band then analyzed by Tricine-SDS-PAGE and autoradiography, as describedin Example III. The granzyme B used for these assays was purified byimmunopurification from human natural killer cell lysates using granzymeB-specific monoclonal antibody (Trapani et al., Biochem. Biophys. Res.Commun. 195:910-920 (1993); Trapani et al., J. Biol. Chem.269:18359-18365 (1994)). The results indicate that granzyme B cleavesproMch6 preferentially at one site to generate a large product ofapproximately 35 kDa (p35) and a small product of approximately 12 kDa(p12).

In comparison with the products of CPP32 processing described in ExampleIII, the large granzyme B product (p35) migrates faster than the largeCPP32 product (p37), indicating that the enzymes cleave at two differentsites. The granzyme B cleavage site is in the N-terminal direction ofthe CPP32 cleavage site and it is most likely to be Asp315 within the³¹²PEPD-A³¹⁶ site (SEQ ID NO: 84).

The location of the granzyme B cleavage sites were confirmed by usingproMch6 mutants as described in Example III. Unlike CPP32, granzyme Bcleaved the Asp330 mutant to generate p35 and p12 products. Whengranzyme B was assayed with the Asp315 mutant, the p35 and p12 productswere absent. This result indicated that granzyme B was unable to processthe ³¹²PEPD-A³¹⁶ site (SEQ ID NO: 84). Granzyme B did cleave the Asp315mutant at the ³²⁷DQLD-A³³¹ site (SEQ ID NO: 85) to generate faint p37and p10 products, albeit inefficiently. A double mutant form of proMch6at Asp315 and Asp330, which was prepared by site-directed mutagenesis asdescribed in Example III, completely blocked processing of proMcH6 bygranzyme B and CPP32. These results show that granzyme B processesproMch6 at Asp315 and Asp330 with preference for Asp315 over Asp330.

EXAMPLE V Activation of ProCPP32 by Granzyme B in a Cell Lysate Lead toProcessing of ProMch6 at Asp315 and Asp330 Simultaneously

This example shows that activation of proCPP32 by granzyme B in celllysate results in cleavage of proMch6 at two sites simultaneously.

As described in Examples III and IV, CPP32 and granzyme B processproMch6 at two different sites. To determine whether granzyme B canprocess proCPP32 into CPP32, followed by simultaneous processing ofproMch6 by granzyme B and CCP32, ³⁵S-labeled proMch6 was mixed with cellextract from 697 lymphocyte cell line and then incubated with granzymeB.

The products of the incubated reaction were analyzed at different timepoints by western blotting to detect activation of endogenous proCPP32.The western blots used a rabbit polyclonal anti-human CPP32 antibody,which was raised against recombinant p20 subunit (amino acids 1-175) ofhuman CPP32. Autoradiography was also used to detect processing ofproMch6. The p20/p19 doublet of mature CPP32 was detected in less than15 minutes, simultaneously with the disappearance of the 32 kDaproCPP32. Subsequently, the p20 product was autocatalytically processedto the p19. In the presence of the DEVD-CHO (SEQ ID NO: 81) inhibitor,only the p20 could be detected due to the inhibition of CPP32 underthese conditions.

Autoradiographic analysis of the same samples revealed a time-dependentprocessing of proMch6 to the p35 (granzyme B product) and p37 (CPP32product) bands. Also detectable was the p12/p10 band. When CPP32 wasinhibited by the DEVD-CHO (SEQ ID NO: 81), only the granzyme B-generatedp35 and p12 bands were detected.

The above results indicate that the p37, p35, p12 and p10 products areproduced in cells undergoing granzyme B-mediated apoptosis. Thisinterpretation is based on the fact that cell lysates contain all thecytoplasmic components necessary for apoptosis (Trapani et al., Biochem.Biophys. Res. Commun. 195:910-920 (1993); Martin, et al., EMBO J.14:5191-5200 (1995); Enari et al., EMBO J. 14:5201-5208 (1995)).Therefore, the mature Mch6 enzyme is derived from the proenzyme bycleavage at Asp315 and Asp330 to generate the large (p35) and small(p10) subunits, as shown in FIG. 3. Due to the lack of evidence ofefficient processing in the prodomain in cell lysates, these resultsindicate that the p35 is the large subunit and p10 is the small subunitof mature Mch6. Moreover, these results further show that activation ofproCPP32 by granzyme B in cell lysate results in cleavage of proMch6 attwo sites simultaneously.

Throughout this application, various publications are referenced withinparentheses. The disclosures of these publications in their entiretiesare hereby incorporated by reference in this application in order tomore fully describe the state of the art to which this inventionpertains.

Although the invention has been described with reference to thedisclosed embodiments, those skilled in the art will readily appreciatethat the specific experiments detailed are only illustrative of theinvention. It should be understood that various modifications can bemade without departing from the spirit of the invention. Accordingly,the invention is limited only by the following claims.

87 1251 base pairs nucleic acid single linear cDNA CDS 1..1251 1 ATG GACGAA GCG GAT CGG CGG CTC CTG CGG CGG TGC CGG CTG CGG CTG 48 Met Asp GluAla Asp Arg Arg Leu Leu Arg Arg Cys Arg Leu Arg Leu 1 5 10 15 GTG GAAGAG CTG CAG GTG GAC CAG CTC TGG GAC GCC CTG CTG AGC AGC 96 Val Glu GluLeu Gln Val Asp Gln Leu Trp Asp Ala Leu Leu Ser Ser 20 25 30 GAG CTG TTCAGG CCC CAT ATG ATC GAG GAC ATC CAG CGG GCA GGC TCT 144 Glu Leu Phe ArgPro His Met Ile Glu Asp Ile Gln Arg Ala Gly Ser 35 40 45 GGA TCT CGG CGGGAT CAG GCC AGG CAG CTG ATC ATA GAT CTG GAG ACT 192 Gly Ser Arg Arg AspGln Ala Arg Gln Leu Ile Ile Asp Leu Glu Thr 50 55 60 CGA GGG AGT CAG GCTCTT CCT TTG TTC ATC TCC TGC TTA GAG GAC ACA 240 Arg Gly Ser Gln Ala LeuPro Leu Phe Ile Ser Cys Leu Glu Asp Thr 65 70 75 80 GGC CAG GAC ATG CTGGCT TCG TTT CTG CGA ACT AAC AGG CAA GCA GCA 288 Gly Gln Asp Met Leu AlaSer Phe Leu Arg Thr Asn Arg Gln Ala Ala 85 90 95 AAG TTG TCG AAG CCA ACCCTA GAA AAC CTT ACC CCA GTG GTG CTC AGA 336 Lys Leu Ser Lys Pro Thr LeuGlu Asn Leu Thr Pro Val Val Leu Arg 100 105 110 CCA GAG ATT CGC AAA CCAGAG GTT CTC AGA CCG GAA ACA CCC AGA CCA 384 Pro Glu Ile Arg Lys Pro GluVal Leu Arg Pro Glu Thr Pro Arg Pro 115 120 125 GTG GAC ATT GGT TCT GGAGGA TTT GGT GAT GTC GGT GCT CTT GAG AGT 432 Val Asp Ile Gly Ser Gly GlyPhe Gly Asp Val Gly Ala Leu Glu Ser 130 135 140 TTG AGG GGA AAT GCA GATTTG GCT TAC ATC CTG AGC ATG GAG CCC TGT 480 Leu Arg Gly Asn Ala Asp LeuAla Tyr Ile Leu Ser Met Glu Pro Cys 145 150 155 160 GGC CAC TGC CTC ATTATC AAC AAT GTG AAC TTC TGC CGT GAG TCC GGG 528 Gly His Cys Leu Ile IleAsn Asn Val Asn Phe Cys Arg Glu Ser Gly 165 170 175 CTC CGC ACC CGC ACTGGC TCC AAC ATC GAC TGT GAG AAG TTG CGG CGT 576 Leu Arg Thr Arg Thr GlySer Asn Ile Asp Cys Glu Lys Leu Arg Arg 180 185 190 CGC TTC TCC TCG CCGCAT TTC ATG GTG GAG GTG AAG GGC GAC CTG ACT 624 Arg Phe Ser Ser Pro HisPhe Met Val Glu Val Lys Gly Asp Leu Thr 195 200 205 GCC AAG AAA ATG GTGCTG GCT TTG CTG GAG CTG GCG CAG CAG GAC CAC 672 Ala Lys Lys Met Val LeuAla Leu Leu Glu Leu Ala Gln Gln Asp His 210 215 220 GGT GCT CTG GAC TGCTGC GTG GTG GTC ATT CTC TCT CAC GGC TGT CAG 720 Gly Ala Leu Asp Cys CysVal Val Val Ile Leu Ser His Gly Cys Gln 225 230 235 240 GCC AGC CAC CTGCAG TTC CCA GGG GCT GTC TAC GGC ACA GAT GGA TGC 768 Ala Ser His Leu GlnPhe Pro Gly Ala Val Tyr Gly Thr Asp Gly Cys 245 250 255 CCT GTG TCG GTCGAG AAG ATT GTG AAC ATC TTC AAT GGG ACC AGC TGC 816 Pro Val Ser Val GluLys Ile Val Asn Ile Phe Asn Gly Thr Ser Cys 260 265 270 CCC AGC CTG GGAGGA AAG CCC AAG CTC TTT TTC ATC CAG GCC TGT GGT 864 Pro Ser Leu Gly GlyLys Pro Lys Leu Phe Phe Ile Gln Ala Cys Gly 275 280 285 GGG GAG CAG AAAGAC CAT GGG TTT GAG GTG GCC TCC ACT TCC CCT GAA 912 Gly Glu Gln Lys AspHis Gly Phe Glu Val Ala Ser Thr Ser Pro Glu 290 295 300 GAC GAG TCC CCTGGC AGT AAC CCC GAG CCA GAT GCC ACC CCG TTC CAG 960 Asp Glu Ser Pro GlySer Asn Pro Glu Pro Asp Ala Thr Pro Phe Gln 305 310 315 320 GAA GGT TTGAGG ACC TTC GAC CAG CTG GAC GCC ATA TCT AGT TTG CCC 1008 Glu Gly Leu ArgThr Phe Asp Gln Leu Asp Ala Ile Ser Ser Leu Pro 325 330 335 ACA CCC AGTGAC ATC TTT GTG TCC TAC TCT ACT TTC CCA GGT TTT GTT 1056 Thr Pro Ser AspIle Phe Val Ser Tyr Ser Thr Phe Pro Gly Phe Val 340 345 350 TCC TGG AGGGAC CCC AAG AGT GGC TCC TGG TAC GTT GAG ACC CTG GAC 1104 Ser Trp Arg AspPro Lys Ser Gly Ser Trp Tyr Val Glu Thr Leu Asp 355 360 365 GAC ATC TTTGAG CAG TGG GCT CAC TCT GAA GAC CTG CAG TCC CTC CTG 1152 Asp Ile Phe GluGln Trp Ala His Ser Glu Asp Leu Gln Ser Leu Leu 370 375 380 CTT AGG GTCGCT AAT GCT GTT TCG GTG AAA GGG ATT TAT AAA CAG ATG 1200 Leu Arg Val AlaAsn Ala Val Ser Val Lys Gly Ile Tyr Lys Gln Met 385 390 395 400 CCT GGTTGC TTT AAT TTC CTC CGG AAA AAA CTT TTC TTT AAA ACA TCA 1248 Pro Gly CysPhe Asn Phe Leu Arg Lys Lys Leu Phe Phe Lys Thr Ser 405 410 415 TAA 1251416 amino acids amino acid linear protein 2 Met Asp Glu Ala Asp Arg ArgLeu Leu Arg Arg Cys Arg Leu Arg Leu 1 5 10 15 Val Glu Glu Leu Gln ValAsp Gln Leu Trp Asp Ala Leu Leu Ser Ser 20 25 30 Glu Leu Phe Arg Pro HisMet Ile Glu Asp Ile Gln Arg Ala Gly Ser 35 40 45 Gly Ser Arg Arg Asp GlnAla Arg Gln Leu Ile Ile Asp Leu Glu Thr 50 55 60 Arg Gly Ser Gln Ala LeuPro Leu Phe Ile Ser Cys Leu Glu Asp Thr 65 70 75 80 Gly Gln Asp Met LeuAla Ser Phe Leu Arg Thr Asn Arg Gln Ala Ala 85 90 95 Lys Leu Ser Lys ProThr Leu Glu Asn Leu Thr Pro Val Val Leu Arg 100 105 110 Pro Glu Ile ArgLys Pro Glu Val Leu Arg Pro Glu Thr Pro Arg Pro 115 120 125 Val Asp IleGly Ser Gly Gly Phe Gly Asp Val Gly Ala Leu Glu Ser 130 135 140 Leu ArgGly Asn Ala Asp Leu Ala Tyr Ile Leu Ser Met Glu Pro Cys 145 150 155 160Gly His Cys Leu Ile Ile Asn Asn Val Asn Phe Cys Arg Glu Ser Gly 165 170175 Leu Arg Thr Arg Thr Gly Ser Asn Ile Asp Cys Glu Lys Leu Arg Arg 180185 190 Arg Phe Ser Ser Pro His Phe Met Val Glu Val Lys Gly Asp Leu Thr195 200 205 Ala Lys Lys Met Val Leu Ala Leu Leu Glu Leu Ala Gln Gln AspHis 210 215 220 Gly Ala Leu Asp Cys Cys Val Val Val Ile Leu Ser His GlyCys Gln 225 230 235 240 Ala Ser His Leu Gln Phe Pro Gly Ala Val Tyr GlyThr Asp Gly Cys 245 250 255 Pro Val Ser Val Glu Lys Ile Val Asn Ile PheAsn Gly Thr Ser Cys 260 265 270 Pro Ser Leu Gly Gly Lys Pro Lys Leu PhePhe Ile Gln Ala Cys Gly 275 280 285 Gly Glu Gln Lys Asp His Gly Phe GluVal Ala Ser Thr Ser Pro Glu 290 295 300 Asp Glu Ser Pro Gly Ser Asn ProGlu Pro Asp Ala Thr Pro Phe Gln 305 310 315 320 Glu Gly Leu Arg Thr PheAsp Gln Leu Asp Ala Ile Ser Ser Leu Pro 325 330 335 Thr Pro Ser Asp IlePhe Val Ser Tyr Ser Thr Phe Pro Gly Phe Val 340 345 350 Ser Trp Arg AspPro Lys Ser Gly Ser Trp Tyr Val Glu Thr Leu Asp 355 360 365 Asp Ile PheGlu Gln Trp Ala His Ser Glu Asp Leu Gln Ser Leu Leu 370 375 380 Leu ArgVal Ala Asn Ala Val Ser Val Lys Gly Ile Tyr Lys Gln Met 385 390 395 400Pro Gly Cys Phe Asn Phe Leu Arg Lys Lys Leu Phe Phe Lys Thr Ser 405 410415 18 base pairs nucleic acid single linear cDNA 3 CTCAACGTAC CAGGAGCC18 19 base pairs nucleic acid single linear cDNA 4 CCTGGGAAAG TAGAGTAGG19 17 base pairs nucleic acid single linear cDNA 5 CAGGAATTCG GCACGAG 1746 amino acids amino acid linear peptide 6 Arg Thr Arg Thr Gly Ser LeuSer His Gly Cys Gln Phe Ile Gln Ala 1 5 10 15 Cys Gly Gly Glu Gln ProGlu Pro Asp Ala Asp Gln Leu Asp Ala Gly 20 25 30 Phe Val Ser Trp Arg AspPro Lys Ser Gly Ser Trp Tyr Val 35 40 45 41 amino acids amino acidlinear peptide 7 Arg Asp Arg Asn Gly Thr Leu Ser His Gly Asp Lys Phe IleGln Ala 1 5 10 15 Cys Gln Gly Asp Asn Val Glu Thr Asp Ser Asn Cys ValSer Tyr Arg 20 25 30 Asn Pro Ala Glu Gly Thr Trp Tyr Ile 35 40 41 aminoacids amino acid linear peptide 8 Lys Asp Arg Gln Gly Thr Leu Thr HisGly Arg Phe Phe Ile Gln Ala 1 5 10 15 Cys Gln Gly Glu Glu Ile Glu AlaAsp Ala Gly Tyr Val Ser Phe Arg 20 25 30 His Val Glu Glu Gly Ser Trp TyrIle 35 40 41 amino acids amino acid linear peptide 9 Gly Val Arg Asn GlyThr Leu Ser His Gly Glu Glu Phe Ile Gln Ala 1 5 10 15 Cys Arg Gly ThrGlu Ile Gln Ala Asp Ser Gly Tyr Tyr Ser Trp Arg 20 25 30 Ser Pro Gly ArgGly Ser Trp Phe Val 35 40 46 amino acids amino acid linear peptide 10Pro Glu Arg Arg Gly Thr Leu Ser His Gly Glu Gly Ile Ile Gln Ala 1 5 1015 Cys Arg Gly Asn Gln Asp Val Val Asp Asn Thr Glu Val Asp Ala Gly 20 2530 Tyr Tyr Ser His Arg Glu Thr Val Asn Gly Ser Trp Tyr Ile 35 40 45 41amino acids amino acid linear peptide 11 Thr Ser Arg Ser Gly Thr Leu SerHis Gly Glu Glu Ile Ile Gln Ala 1 5 10 15 Cys Arg Gly Thr Glu Ile GluThr Asp Ser Gly Tyr Tyr Ser Trp Arg 20 25 30 Asn Ser Lys Asp Gly Ser TrpPhe Ile 35 40 41 amino acids amino acid linear peptide 12 Pro Thr ArgAsn Gly Thr Leu Ser His Gly Glu Glu Phe Val Gln Ala 1 5 10 15 Cys ArgGly Glu Arg Asp Ser Val Asp Gly Gln Tyr Val Ser Trp Arg 20 25 30 Asn SerAla Arg Gly Ser Trp Phe Ile 35 40 46 amino acids amino acid linearpeptide 13 Pro Arg Arg Thr Gly Ala Met Ser His Gly Ile Arg Ile Ile GlnAla 1 5 10 15 Cys Arg Gly Asp Ser Trp Phe Lys Asp Ser Phe Glu Asp AspAla Asp 20 25 30 Asn Val Ser Trp Arg His Pro Thr Met Gly Ser Val Phe Ile35 40 45 46 amino acids amino acid linear peptide 14 Pro Pro Arg Asn GlyAla Met Ser His Gly Ile Leu Ile Val Gln Ala 1 5 10 15 Cys Arg Gly AlaAsn Trp Val Lys Asp Ser Leu Glu Glu Asp Ala His 20 25 30 Asn Val Ser TrpArg Asp Ser Thr Met Gly Ser Ile Phe Ile 35 40 45 46 amino acids aminoacid linear peptide 15 Pro Ala Arg Asn Gly Ala Met Ser His Gly Ile LeuIle Val Gln Ala 1 5 10 15 Cys Arg Gly Glu Lys Trp Val Arg Asp Ser LeuGlu Ala Asp Ser His 20 25 30 Asn Val Ser Trp Arg Asp Arg Thr Arg Gly SerIle Phe Ile 35 40 45 46 amino acids amino acid linear peptide 16 Glu PheArg Ser Gly Gly Leu Ser His Gly Val Glu Phe Ile Gln Ala 1 5 10 15 CysArg Gly Asp Glu Asp Gln Gln Asp Gly Glu Glu Ser Asp Ala Gly 20 25 30 ThrAla Ala Met Arg Asn Thr Lys Arg Gly Ser Trp Tyr Ile 35 40 45 6 aminoacids amino acid single linear peptide 17 Arg Thr Arg Thr Gly Ser 1 5 6amino acids amino acid linear peptide 18 Leu Ser His Gly Cys Gln 1 5 9amino acids amino acid linear peptide 19 Phe Ile Gln Ala Cys Gly Gly GluGln 1 5 5 amino acids amino acid linear peptide 20 Pro Glu Pro Asp Ala 15 5 amino acids amino acid linear peptide 21 Asp Gln Leu Asp Ala 1 5 15amino acids amino acid linear peptide 22 Gly Phe Val Ser Trp Arg Asp ProLys Ser Gly Ser Trp Tyr Val 1 5 10 15 6 amino acids amino acid linearpeptide 23 Arg Asp Arg Asn Gly Thr 1 5 6 amino acids amino acid linearpeptide 24 Leu Ser His Gly Asp Lys 1 5 9 amino acids amino acid linearpeptide 25 Phe Ile Gln Ala Cys Gln Gly Asp Asn 1 5 5 amino acids aminoacid linear peptide 26 Val Glu Thr Asp Ser 1 5 15 amino acids amino acidlinear peptide 27 Asn Cys Val Ser Tyr Arg Asn Pro Ala Glu Gly Thr TrpTyr Ile 1 5 10 15 6 amino acids amino acid linear peptide 28 Lys Asp ArgGln Gly Thr 1 5 6 amino acids amino acid linear peptide 29 Leu Thr HisGly Arg Phe 1 5 9 amino acids amino acid linear peptide 30 Phe Ile GlnAla Cys Gln Gly Glu Glu 1 5 5 amino acids amino acid linear peptide 31Ile Glu Ala Asp Ala 1 5 15 amino acids amino acid linear peptide 32 GlyTyr Val Ser Phe Arg His Val Glu Glu Gly Ser Trp Tyr Ile 1 5 10 15 6amino acids amino acid linear peptide 33 Gly Val Arg Asn Gly Thr 1 5 6amino acids amino acid linear peptide 34 Leu Ser His Gly Glu Glu 1 5 9amino acids amino acid linear peptide 35 Phe Ile Gln Ala Cys Arg Gly ThrGlu 1 5 5 amino acids amino acid linear peptide 36 Ile Gln Ala Asp Ser 15 15 amino acids amino acid linear peptide 37 Gly Tyr Tyr Ser Trp ArgSer Pro Gly Arg Gly Ser Trp Phe Val 1 5 10 15 6 amino acids amino acidlinear peptide 38 Pro Glu Arg Arg Gly Thr 1 5 6 amino acids amino acidlinear peptide 39 Leu Ser His Gly Glu Gly 1 5 9 amino acids amino acidlinear peptide 40 Ile Ile Gln Ala Cys Arg Gly Asn Gln 1 5 5 amino acidsamino acid linear peptide 41 Asp Val Val Asp Asn 1 5 5 amino acids aminoacid linear peptide 42 Thr Glu Val Asp Ala 1 5 15 amino acids amino acidlinear peptide 43 Gly Tyr Tyr Ser His Arg Glu Thr Val Asn Gly Ser TrpTyr Ile 1 5 10 15 6 amino acids amino acid linear peptide 44 Thr Ser ArgSer Gly Thr 1 5 6 amino acids amino acid linear peptide 45 Leu Ser HisGly Glu Glu 1 5 9 amino acids amino acid linear peptide 46 Ile Ile GlnAla Cys Arg Gly Thr Glu 1 5 5 amino acids amino acid linear peptide 47Ile Glu Thr Asp Ser 1 5 15 amino acids amino acid linear peptide 48 GlyTyr Tyr Ser Trp Arg Asn Ser Lys Asp Gly Ser Trp Phe Ile 1 5 10 15 6amino acids amino acid linear peptide 49 Pro Thr Arg Asn Gly Thr 1 5 6amino acids amino acid linear peptide 50 Leu Ser His Gly Glu Glu 1 5 9amino acids amino acid linear peptide 51 Phe Val Gln Ala Cys Arg Gly GluArg 1 5 5 amino acids amino acid linear peptide 52 Asp Ser Val Asp Gly 15 15 amino acids amino acid linear cDNA 53 Gln Tyr Val Ser Trp Arg AsnSer Ala Arg Gly Ser Trp Phe Ile 1 5 10 15 6 amino acids amino acidlinear peptide 54 Pro Arg Arg Thr Gly Ala 1 5 6 amino acids amino acidlinear peptide 55 Met Ser His Gly Ile Arg 1 5 9 amino acids amino acidlinear peptide 56 Ile Ile Gln Ala Cys Arg Gly Asp Ser 1 5 5 amino acidsamino acid linear peptide 57 Trp Phe Lys Asp Ser 1 5 5 amino acids aminoacid linear peptide 58 Phe Glu Asp Asp Ala 1 5 15 amino acids amino acidlinear peptide 59 Asp Asn Val Ser Trp Arg His Pro Thr Met Gly Ser ValPhe Ile 1 5 10 15 6 amino acids amino acid linear peptide 60 Pro Pro ArgAsn Gly Ala 1 5 6 amino acids amino acid linear peptide 61 Met Ser HisGly Ile Leu 1 5 9 amino acids amino acid linear peptide 62 Ile Val GlnAla Cys Arg Gly Ala Asn 1 5 5 amino acids amino acid linear peptide 63Trp Val Lys Asp Ser 1 5 5 amino acids amino acid linear peptide 64 LeuGlu Glu Asp Ala 1 5 15 amino acids amino acid linear peptide 65 His AsnVal Ser Trp Arg Asp Ser Thr Met Gly Ser Ile Phe Ile 1 5 10 15 6 aminoacids amino acid linear peptide 66 Pro Ala Arg Asn Gly Ala 1 5 6 aminoacids amino acid linear peptide 67 Met Ser His Gly Ile Leu 1 5 9 aminoacids amino acid linear peptide 68 Ile Val Gln Ala Cys Arg Gly Glu Lys 15 5 amino acids amino acid linear peptide 69 Trp Val Arg Asp Ser 1 5 5amino acids amino acid linear peptide 70 Leu Glu Ala Asp Ser 1 5 15amino acids amino acid linear peptide 71 His Asn Val Ser Trp Arg Asp ArgThr Arg Gly Ser Ile Phe Ile 1 5 10 15 6 amino acids amino acid linearpeptide 72 Glu Phe Arg Ser Gly Gly 1 5 6 amino acids amino acid linearpeptide 73 Leu Ser His Gly Val Glu 1 5 9 amino acids amino acid linearpeptide 74 Phe Ile Gln Ala Cys Arg Gly Asp Glu 1 5 5 amino acids aminoacid linear peptide 75 Asp Gln Gln Asp Gly 1 5 5 amino acids amino acidlinear peptide 76 Glu Glu Ser Asp Ala 1 5 15 amino acids amino acidlinear peptide 77 Gly Thr Ala Ala Met Arg Asn Thr Lys Arg Gly Ser TrpTyr Ile 1 5 10 15 5 amino acids amino acid linear peptide 78 Gln Ala CysGly Gly 1 5 5 amino acids amino acid linear peptide 79 Gln Ala Cys ArgGly 1 5 5 amino acids amino acid linear peptide 80 Gln Ala Cys Gln Gly 15 4 amino acids amino acid linear peptide Peptide /note= “Amino Acid isbonded to an aldehyde at the C-terminal.” 81 Asp Glu Val Asp 1 4 aminoacids amino acid linear peptide Peptide /note= “Amino acid is bonded toan aminomethylcoumarin at the C-terminal.” 82 Asp Glu Val Asp 1 4 aminoacids amino acid linear peptide Peptide /note= “Amino acid is bonded toaminomethylcoumarin at the C-terminal.” 83 Tyr Val Ala Asp 1 5 aminoacids amino acid linear peptide 84 Pro Glu Pro Asp Ala 1 5 5 amino acidsamino acid linear peptide 85 Asp Gln Leu Asp Ala 1 5 5 amino acids aminoacid linear peptide 86 Asp Glu Val Asp Gly 1 5 5 amino acids amino acidlinear peptide 87 Asp Val Val Asp Asn 1 5

What is claimed is:
 1. An antibody that specifically binds a Mch6polypeptide, said Mch6 polypeptide comprising the amino acid sequenceshown in SEQ ID NO:2, or an enzymatically active fragment thereof. 2.The antibody of claim 1, wherein the enzymatically active fragmentcomprises the catalytic domain set forth in SEQ ID NO:78.
 3. An antibodythat specifically binds an isolated large subunit of an Mch6polypeptide, said polypeptide comprising amino acids 1 to 315 or aminoacids 1 to 330 as shown in SEQ ID NO:2, or an enzymatically activefragment thereof.
 4. An antibody that specifically binds an isolatedsmall subunit of Mch6 polypeptide, said polypeptide comprising aminoacids 316 to 416 or 331 to 416 as shown in SEQ ID NO:2.